Oral drug delivery compositions and methods

ABSTRACT

The present invention relates to an oral drug delivery system, and in particular to modified amino acids and modified amino acid derivatives for use as a delivery system of sensitive agents such as bioactive peptides. The modified amino acids and derivatives can form non-covalent mixtures with active biological agents and in an alternate embodiment can releasably carry active agents. Modified amino acids can also form drug containing microspheres. These mixtures are suitable for oral administration of biologically active agents to animals. Methods for the preparation of such amino acids are also disclosed.

This application is a continuation of U.S. application Ser. No.09/862,063, filed now May 21, 2001; now U.S. Pat. No. 6,461,643, whichis a continuation of U.S. application Ser. No. 09/346,971, filed Jul. 2,1999, now abandoned; which is a continuation of U.S. application Ser.No. 08/438,644, filed May 10, 1995, now U.S. Pat. No. 5,958,457; whichis a continuation-in-part of (a) U.S. application Ser. No. 08/335,147,filed Oct. 25, 1994, now abandoned; (b) PCT Application Serial No.PCT/US94/04560 filed Apr. 22, 1994; which is a continuation-in-part ofU.S. application Ser. No. 08/051,019, filed Apr. 22, 1993, now U.S. Pat.No. 5,451,410; and of U.S. application Ser. No. 08/205,511, filed Mar.2, 1994, now U.S. Pat. No. 5,792,451; and (c) U.S. application Ser. No.08/231,622, filed Apr. 22, 1994, now U.S. Pat. No. 5,629,020. Theseprior applications are hereby incorporated herein by reference, in theirentirety.

FIELD OF THE INVENTION

The present invention relates to compositions suitable for oral drugdelivery, and in particular to compositions in which modified aminoacids and modified amino acid derivatives are used as carriers forsensitive agents such as bioactive peptides and the like. The modifiedamino acids or derivatives can form non-covalent mixtures withbiologically-active agents which are suitable for oral administration toanimals. Methods for the preparation and for the administration of suchcompositions are also disclosed.

BACKGROUND OF THE INVENTION

Conventional means for delivering biologically-active agents, including,but not limited to, pharmaceutical and therapeutic agents, to animalsare often severely limited by chemical barriers and physical barriersimposed by the body. Oral delivery of many biologically-active agentswould be the route of choice if not for chemical and physico-chemicalbarriers such as the extreme and varying pH in the gastro-intestinal(GI) tract, exposure to powerful digestive enzymes, and theimpermeability of gastro-intestinal membranes to the active agent. Amongthe numerous agents which are not typically suitable for oraladministration are biologically active peptides such as calcitonin andinsulin. Examples of other compounds which are affected by thesephysico-chemical barriers are polysaccharides and particularlymucopolysaccharides, including, but not limited to, heparin;heparinoids; antibiotics; and other organic substances. These agents arerapidly destroyed in the gastro-intestinal tract by acid hydrolysis,enzymes, or the like.

Prior methods for orally administering vulnerable pharmacological agentshave relied on the co-administration of adjuvants (e.g., resorcinols andnon-ionic surfactants such as polyoxyethylene oleyl ether andn-hexadecyl polyethylene ether) to increase artificially thepermeability of the intestinal walls; and on the co-administration ofenzymatic inhibitors (e.g., pancreatic trypsin inhibitor,diisopropylfluorophosphate (DFF) and trasylol) to inhibit enzymaticdegradation. Liposomes have also been described as drug delivery systemsfor insulin and heparin. See, for instance, U.S. Pat. No. 4,239,754;Patel et al. (1976) FEBS Letters Vol. 62, page 60; and Hashimoto et al.(1979) Endocrinol. Japan, Vol. 26, page 337. However, broad spectrum useof the aforementioned drug delivery systems is precluded for reasonsincluding: (1) the need to use toxic amounts of adjuvants or inhibitors;(2) the lack of suitable low MW cargoes; (3) the poor stability andinadequate shelf life of the systems; (4) the difficulties inmanufacturing the systems; (5) the failure of the systems to protect theactive ingredient; and (6) the failure of the systems to promoteabsorption of the active agent.

More recently, microspheres of artificial polymers or proteinoids ofmixed amino acids have been described for delivery of pharmaceuticals.For example, U.S. Pat. No. 4,925,673 describes drug containingmicrosphere constructs as well as methods for their preparation and use.These proteinoid microspheres are useful for delivery of a number ofactive agents.

There is still a need in the art for simple and inexpensive deliverysystems which are easily prepared and which can deliver a broad range ofbiologically-active agents.

SUMMARY OF THE INVENTION

Compositions for orally delivering biologically-active agentsincorporating modified amino acids, amino acid derivatives, peptides andpeptide derivatives as carriers are provided. These compositionscomprise

-   -   (A) at least one biologically-active agent; and    -   (B) at least one carrier comprising        -   (a) (i) at least one acylated aldehyde of an amino acid,            -   (ii) at least one acylated ketone of an amino acid,            -   (iii) at least one acylated aldehyde of a peptide,            -   (iv) at least one acylated ketone of a peptide, or            -   (v) any combination of (a)(i), (a)(ii), (a)(iii) and                (a)(iv);        -   (b) (i) carboxymethyl-phenylalanine-leucine,            -   (ii) 2-carboxy-3-phenylpropionyl-leucine,            -   (iii) 2-benzylsuccinic acid,            -   (iv) an actinonin, or            -   (v) a compound having the formula:                Ar—Y—(R¹)_(n)—OH            -    wherein: Ar is a substituted or unsubstituted phenyl or                naphthyl;            -   Y is            -    or —SO₂—;            -   R¹ is            -    wherein:            -   R³ is C₁ to C₂₄ alkyl, C₁ to C₂₄ alkenyl, phenyl,                naphthyl, (C₁ to C₁₀ alkyl)phenyl, (C₁ to C₁₀                alkenyl)phenyl, (C₁ to C₁₀ alkyl) naphthyl, (C₁ to C₁₀                alkenyl) naphthyl, phenyl(C₁ to C₁₀ alkyl), phenyl(C₁ to                C₁₀ alkenyl), naphthyl(C₁ to C₁₀ alkyl) and naphthyl(C₁                to C₁₀ alkenyl);            -   R³ is optionally substituted with C₁ to C₄ alkyl, C₁ to                C₄ alkenyl, C₁ to C₄ alkoxy, —OH, —SH, —CO₂R⁵,                cycloalkyl, cycloalkenyl, heterocyclic, aryl, alkaryl,                heteroaryl or heteroalkaryl or any combination thereof;            -   R⁵ is hydrogen, C₁ to C₄ alkyl or C₁ to C₄ alkenyl;            -   R³ is optionally interrupted by oxygen, nitrogen, sulfur                or any combination thereof; and            -   R⁴ is hydrogen, C₁ to C₄ alkyl or C₁ to C₄ alkenyl;            -   and n is from 1 to about 5;            -   (vi) or any combination of (b)(i), (b)(ii),            -   (b)(iii), (b)(iv) and (b)(v); or        -   (c) a combination of (a) and (b).            Also contemplated is a method for preparing these            compositions which comprises mixing at least one            biologically active agent, with at least one carrier as            described above, and optionally, a dosage vehicle.

In an alternative embodiment, these non-toxic carriers are orallyadministered to animals as part of a delivery system by blending ormixing the carriers with a biologically active agent prior toadministration. The carriers may also form microspheres in the presenceof the active agent. The microspheres containing the active agent arethen orally administered. Also contemplated by the present invention aredosage unit forms that include these compositions.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphic illustration of the results of oral gavage testingin rats using salmon calcitonin with acetyl phenylalanine aldehyde,carbomethoxyPhe-Leu-OH, and acetyl-Phe-Leu-Leu-Arg aldehyde carriers.

FIG. 2 is a graphic illustration of the results of oral gavage testingin rats using salmon calcitonin with acetylleucine aldehyde andacetylphenylalanine aldehyde carriers.

FIG. 3 is a graphic illustration of the results of oral gavage testingin rats using salmon calcitonin with acetylphenylalanine aldehyde andcarbomethoxyPhe-Leu-OH carriers.

FIG. 4 is a graphic illustration of the results of oral gavage testingin rats using salmon calcitonin with acetylphenylalanine aldehyde,acetylLeu-Leu-Arg aldehyde and carbomethoxyPhe-Leu-OH carriers.

FIG. 5 is a graphic illustration of the results of intraduodenalinjection testing in rats using salmon calcitonin withacetylphenylalanine aldehyde and 4-(phenylsulfonamido)-4-phenylbutyricacid carriers.

FIG. 6 is a graphic illustration of the results of oral gavage testingin rats using salmon calcitonin with acetylphenylalanine aldehyde,N-acetyllysinone, and acetyl-Leu aldehyde carriers.

FIG. 7 is a graphic illustration of the results of intraduodenalinjection testing in rats using salmon calcitonin withacetylphenylalanine aldehyde carrier in aqueous ethanol, dimethylsulfoxide (DMSO), and olive oil dosing vehicles, and in a DMSO dosingvehicle alone.

FIG. 8 is a graphic illustration of the results of oral gavage testingin rats using salmon calcitonin with cyclohexanoyl-phenylalaninealdehyde carrier.

FIG. 9 is a graphic illustration of rat serum calcium levels after oraladministration of two dosage levels of a modified amino acid microspherepreparation containing salmon calcitonin and a soluble modified aminoacid preparation containing salmon calcitonin after pre-dosing with asodium bicarbonate solution.

FIG. 10 is a graphic illustration of the results of oral gavage testingin rats using salmon calcitonin with acetyl-Phe aldehyde, actinonin, andcarbomethoxy-Phe-Leu-OH carriers.

FIG. 11 is a graphic illustration of the results of oral gavage testingin rats using salmon calcitonin with4-(phenylsulfonamido)-4-phenylbutyric acid carrier.

FIG. 12 is a graphic illustration of the results of oral gavage testingin rats using salmon calcitonin with 3-(phenylsulfonamido)benzoic acidand 4-(phenylsulfonamido)-hippuric acid carriers.

FIG. 13 is a graphic illustration of the results of oral gavage testingin rats using salmon calcitonin with4-(phenylsulfonamido)-4-phenylbutyric acid and4-(phenylsulfonamido)benzoic acid carriers.

FIG. 14 is a graphic illustration of the results of oral gavage testingin rats using salmon calcitonin with4-(phenylsulfonamido)-4-phenylbutyric acid and4-(phenylsulfonamido)phenylacetic acid carriers.

FIG. 15 is a graphic illustration of the results of oral gavage testingin rats using interferon α2b (rhIFN) with4-(phenylsulfonamido)-4-phenylbutyric acid carrier.

FIG. 16 is a graphic illustration of the results of oral gavage testingin rats using interferon α2b with 4-(phenylsulfonamidomethyl)benzoicacid carrier.

FIG. 17 is a graphic illustration of the results of oral gavage testingin rats using interferon α2b with 4-(phenylsulfonamido)phenylacetic acidas carrier.

FIG. 18 is a graphic illustration of the results of oral gavage testingin rats using interferon α2b with 4-(phenylsulfonamido)hippuric acidcarrier.

FIGS. 19 and 20 are graphic illustrations of the results of oral gavagetesting in hypophysectomized rats using growth hormone alone and at twodosage levels with 4-(phenylsulfonamido)-4-phenylbutyric acid carrier.

FIG. 21 is a graphic illustration of the results of oral gavage testingin normal rats using growth hormone with4-(phenylsulfonamido)-4-phenylbutyric acid carrier.

FIG. 22 is a graphic illustration of the results of oral gavage testingin rats using disodium cromoglycate with4-(phenylsulfonamido)-4-phenylbutyric acid as carrier.

DETAILED DESCRIPTION OF THE INVENTION

Amino acids and amino acid derivatives, in modified form, may be used todeliver orally sensitive biologically-active agents, including, but notlimited to, hormones such as calcitonin, insulin, and polysaccharidessuch as heparin, which would not be considered orally administrable forvarious reasons. Insulin, for example is sensitive to the denaturingconditions of the gastro-intestinal (GI) tract. Also, heparin, by virtueof its charge and hydrophilic nature, is not readily absorbed from thegastro-intestinal tract. In contrast to the modified amino acids andmodified amino acid derivatives of the present invention, unmodifiedfree amino acids do not provide protection against degradation in the GItract for labile bioactive agents.

The compositions of the subject invention are useful for administeringbiologically-active agents to any animals such as birds; mammals, suchas primates and particularly humans; and insects.

Other advantages provided by the present invention include the use ofreadily available and inexpensive starting materials in cost-effectivemethods for preparing and isolating modified amino acid derivatives.These methods are simple to perform and are amenable to industrialscale-up for commercial production.

Biologically-active agents suitable for use with carriers disclosedherein include, but are not limited to, peptides, and particularly smallpeptide hormones, which by themselves do not pass or only pass slowlythrough the gastro-intestinal mucosa and/or are susceptible to chemicalcleavage by acids and enzymes in the gastro-intestinal tract;polysaccharides and particularly mixtures of muco-polysaccharides;carbohydrates; lipids; or any combination thereof. Examples include, butare not limited to, human growth hormone; bovine growth hormone; growthhormone releasing hormone; interferons; interleukin-I; insulin; heparin,and particularly low molecular weight heparin; calcitonin;erythropoietin; atrial naturetic factor; antigens; monoclonalantibodies; somatostatin; adrenocorticotropin; gonadotropin releasinghormone; oxytocin; vasopressin; cromolyn sodium (sodium or disodiumcromoglycate); vancomycin; desferrioxamine (DFO); or any combinationthereof.

Additionally the carriers of the present invention can be used todeliver other active agents such as pesticides and the like.

The term amino acid as used herein includes any carboxylic acid havingat least one free amine group including naturally occurring andsynthetic amino acids. The preferred amino acids are ∝-amino acids, andpreferably are naturally occurring ∝-amino acids although non-α-aminoacids are useful as well.

Poly amino acids as used herein refers to peptides or two or more aminoacids linked by a bond formed by other groups which can be linked, e.g.,an ester, anhydride or an anhydride linkage.

The term peptide is meant to include two or more amino acids joined by apeptide bond. Peptides can vary in length from dipeptides with 2 to polypeptides with several hundred amino acids. See Chambers BiologicalDictionary, editor Peter M. B. Walker, Cambridge, England: ChambersCambridge, 1989, page 215. The peptides most useful in the practice ofthe present invention include di-peptides, tri-peptides, tetra-peptides,and penta-peptides. The preferred peptides are di-peptides,tri-peptides. Peptides can be homo- or hetero-peptides and can includenatural amino acids, synthetic amino acids, or any combination thereof.

The term amino acid derivatives and peptide derivatives as used hereinare meant to include amino acid aldehydes or ketones and/or peptidealdehydes or ketones where the —COOH group has been converted to aketone or aldehyde.

The terms modified amino acids, peptides, and derivatives thereof aremeant to include amino acids, amino acid derivatives, peptides andpeptide derivatives which have been modified as described below byacylating or sulfonating at least one free amine group, with anacylating or sulfonating agent which reacts with at least one of thefree amine groups present.

The preferred naturally occurring amino acids for use in the presentinvention as amino acids or components of a peptide are alanine,arginine, asparagine, aspartic acid, citrulline, cysteine, cystine,glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine,ornithine, phenylalanine, proline, serine, threonine, tryptophan,tyrosine, valine, hydroxy proline, γ-carboxyglutamate, orO-phosphoserine. The most preferred amino acids are arginine, leucine,lysine, phenylalanine, tyrosine and valine.

The preferred non-naturally occurring amino acids for use in the presentinvention as amino acids or components of a peptide are β-alanine,phenylglycine, α-aminobutyric acid, γ-amino butyric acid,4-(4-aminophenyl)butyric acid, α-amino isobutyric acid, ε-aminocaproicacid, 7-aminoheptanoic acid, β-aspartic acid, aminobenzoic acid,(aminomethyl)benzoic acid, aminophenylacetic acid, aminohippuric acid,γ-glutamic acid, cysteine(ACM), ε-lysine, ε-lysine (A-Fmoc), methioninesulfone, norleucine, norvaline, ornithine, d-ornithine,p-nitrophenylalanine, hydroxy proline, and thioproline.

The amino acids useful in the practice of the subject invention have theformula:HN(R⁴)—(R²)_(n)—OH

R² has the formula

wherein R³ is C₁ to C₂₄ alkyl, C₁ to C₂₄ alkenyl, phenyl, naphthyl, (C₁to C₁₀ alkyl)-phenyl, (C₁ to C₁₀ alkenyl)phenyl, (C₁ to C₁₀ alkyl)naphthyl, (C₁ to C₁₀ alkenyl)naphthyl, phenyl (C₁ to C₁₀ alkyl), phenyl(C₁ to C₁₀ alkenyl), naphthyl(C₁ to C₁₀ alkyl) and naphthyl (C₁ to C₁₀alkenyl);

optionally R³ is substituted with C₁ to C₄ alkyl, C₁ to C₄alkenyl, C₁ toC₄ alkoxy, —OH, —SH and —CO₂R⁵ or any combination thereof;

R⁵ is hydrogen, C₁ to C₄ alkyl or C₁ to C₄ alkenyl;

R³ is optionally interrupted by oxygen, nitrogen, sulfur or anycombination thereof; and

R⁴ is hydrogen, C₁ to C₄ alkyl or C₁ to C₄ alkenyl.

The phenyl or naphthyl groups can be optionally substituted. Suitablebut non-limiting examples of substitutents are C₁ to C₆ alkyl, C₁ to C₆alkenyl, alkoxy having from 1 to 6 carbon atoms, hydroxy, thio, or CO₂R⁶wherein R⁶ is hydrogen, C₁ to C₆ alkyl, C₁ to C₆ alkenyl.

The amino acid derivatives or peptide derivatives of the presentinvention can be readily prepared by reduction of amino acid esters orpeptide esters with an appropriate reducing agent. For example, aminoacid aldehydes or peptide aldehydes can be prepared as described in anarticle by R. Chen et al., Biochemistry, 1979, 18, 921–926. Amino acidor peptide ketones can be prepared by the procedure described in OrganicSyntheses, Col. Vol. IV, Wiley, (1963), pages 5–6. Amino acids,peptides, amino acid esters, peptide esters, and other necessaryreagents to prepare these derivatives are readily available from anumber of commercial sources such as Aldrich Chemical Co. (Milwaukee,Wis., USA); Sigma Chemical Co. (St. Louis, Mo., USA); and Fluka ChemicalCorp. (Ronkonkoma, N.Y., USA).

The amino acids and peptides are modified by acylating or sulfonating atleast one free amine group, with an acylating or sulfonating agent whichreacts with at least one of the free amine groups present. Suitable, butnon-limiting, examples of agents useful for modifying amino acids orpeptides useful in practicing the present invention include acylatingand sulfonating agents having the formula

or R⁷—SO₂—X wherein R⁷ is alkyl or alkenyl, preferably having from 1 to20 carbon atoms, or aromatic preferably having from 6 to 20 carbonatoms.

The R⁷ group can be substituted or unsubstituted, The preferredsubstitutents include C₁ to C₄ alkyl, C₁ to C₄ alkenyl, C₁ to C₄ alkoxy,CO₂R⁸ wherein R⁸ is hydrogen, C₁ to C₄ alkyl or C₁ to C₄ alkenyl.

Preferably, R⁷ is methyl, ethyl, phenyl, benzyl or naphthyl. Morepreferably, R⁷ is phenyl, or acetyl. X is a leaving group. In a reactionin which the substrate molecule becomes cleaved, part of it (the partnot containing the carbon) is usually called the leaving group. SeeAdvanced Organic Chemistry, 2d edition, Jerry March, New York:McGraw-Hill Book (1977), page 187, Typical leaving groups include, butare not limited to, halogens such as chlorine, bromine and iodine.

Examples of the acylating and sulfonating agents for amino acids andpeptides include, but are not limited to, acyl halides such as acetylchloride, propyl chloride, benzoyl chloride, hippuryl chloride and thelike; sulfonyl halides such as benzene sulfonyl chloride, andanhydrides, such as acetic anhydride, propyl anhydride, benzoicanhydride, hippuric anhydride and the like. The preferred acylating andsulfonating agents are benzoyl chloride, benzene sulfonyl chloride, andhippuryl chloride.

The modified acid compounds have the formula:Ar—Y—(R¹)_(n)—OHwherein Ar is a substituted or unsubstituted phenyl or naphthyl;

Y is

or —SO₂—, R¹ has the formula

wherein:

R³ is C₁ to C₂₄ alkyl, C₁ to C₂₄ alkenyl, phenyl, naphthyl, (C₁ to C₁₀alkyl) phenyl, (C₁ to C₁₀ alkenyl) phenyl, (C₁ to C₁₀ alkyl) naphthyl,(C₁ to C₁₀ alkenyl) naphthyl, phenyl (C₁ to C₁₀ alkyl), phenyl (C₁ toC₁₀ alkenyl), naphthyl (C₁ to C₁₀ alkyl) and naphthyl (C₁ to C₁₀alkenyl);

R³ is optionally substituted with C₁ to C₄ alkyl, C₁ to C₄ alkenyl, C₁to C₄ alkoxy, —OH, —SH and —CO₂R⁵ or any combination thereof;

R⁵ is hydrogen, C₁ to C₄ alkyl or C₁ to C₄ alkenyl;

R³ is optionally interrupted by oxygen, nitrogen, sulfur or anycombination thereof; and

R⁴ is hydrogen, C₁ to C₄ alkyl or C₁ to C₄ alkenyl.

The amino acid derivatives and peptide derivatives are modified byacylating at least one free amine group, with an acylating agent whichreacts with at least one of the free amine groups present. Suitable, butnon-limiting, examples of acylating agents useful for modifying aminoacid derivatives and peptide derivatives useful in practicing thepresent invention include acid chloride acylating agents having theformula

or wherein:

R⁹ is alkyl or alkenyl, preferably having from 1 to 20 carbon atoms,cycloalkyl or cycloalkenyl, preferably having from 1 to 20 carbon atoms,or aromatic preferably having from 6 to 20 carbon atoms. The R⁹ groupcan be substituted or unsubstituted, The preferred substituents includeC₁ to C₄ alkyl, C₁ to C₄ alkenyl, C₁ to C₄ alkoxy, CO₂R¹⁰ wherein R¹⁰ ishydrogen, C₁ to C₄ alkyl or C₁ to C₄ alkenyl. Preferably, R⁹ is methyl,ethyl, cyclohexyl, cyclopentyl, cycloheptyl, phenyl, benzyl or naphthyl.More preferably, R⁹ is phenyl, cyclohexyl cyclopentyl, cycloheptyl, oracetyl. X is a leaving group. Typical leaving groups include, but arenot limited to, halogens such as chlorine, bromine and iodine.

Examples of the acylating agents for amino acid derivatives and peptidederivatives include, but are not limited to, acyl halides such as acetylchloride, propyl chloride, cyclohexanoyl chloride, cyclopentanoylchloride, and cycloheptanoyl chloride, benzoyl chloride, hippurylchloride and the like; and anhydrides, such as acetic anhydride, propylanhydride, cyclohexanoic anhydride, benzoic anhydride, hippuricanhydride and the like. The preferred acylating agents are benzoylchloride, benzene sulfonyl chloride, hippuryl chloride, acetyl chloride,cyclohexanoyl chloride, cyclopentanoyl chloride, and cycloheptanoylchloride.

The amine groups can also be modified by the reaction of a carboxylicacid with coupling agents such as dicyclohexylcarbodiimide and the like.In a peptide one or more of the amino acids may be derivatized (analdehyde or a ketone) and/or modified (acylated).

Also suitable as a carrier alone or in combination with the modifiedamino acid or peptide derivatives are the carbomethoxy modified aminoacids carboxy-methyl-phenylalanine-leucine,2-carboxy-3-phenylpropionyl-leucine, 2-benzylsuccinic acid and anactinonin. The actinonin compounds include actinonin or epiactinonin andderivatives thereof. These compounds have the formulas below:

Derivatives of these compounds are disclosed in U.S. Pat. No. 5,206,384.Actinonin derivatives have the formula:

wherein R¹¹ is sulfoxymethyl or carboxyl or a substituted carboxy groupselected from carboxamide, hydroxyaminocarbonyl and alkoxycarbonylgroups; and R¹² is hydroxyl, alkoxy, hydroxyamino or sulfoxyamino group.

The modified amino acid derivatives or peptide derivatives can bereadily prepared and modified by methods known to those skilled in theart. For example, the modified amino acid derivatives of the presentinvention may be prepared by reacting a single amino acid derivative orpeptide derivative or mixtures of two or more amino acid or peptidederivatives, with an acylating agent or an amine modifying agent whichreacts with free amino moieties present in the derivatives to formamides. The amino acid or peptide can be modified and subsequentlyderivatized, derivatized and subsequently modified, or simultaneouslymodified and derivatized. Protecting groups may be used to avoidunwanted side reactions as would be known to those skilled in the art.

The modified amino acids and modified amino acid derivatives of thepresent invention may also be prepared by reacting single amino acids,mixtures of two or more kinds of amino acids, or amino acid esters withan amine modifying agent which reacts with free amino moieties presentin the amino acids to form amides or sulfonamides. Amino acids and aminoacid esters are readily available from a number of commercial sourcessuch as Aldrich Chemical Co. (Milwaukee, Wis., U.S.A.); Sigma ChemicalCo. (St. Louis, Mo., USA); and Fluka Chemical Corp. (Ronkonkoma, N.Y.,USA).

For example, the amino acids can be dissolved in aqueous alkalinesolution of a metal hydroxide, e.g., sodium or potassium hydroxide, andheated at a temperature ranging between about 5° C. and about 70° C.,preferably between about 10° C. and about 40° C., for a period rangingbetween about 1 hour and about 4 hours, preferably about 2.5 hours. Theamount of alkali employed per equivalent of NH₂ groups in the aminoacids generally ranges between about 1.25 and about 3 mmole, preferablybetween about 1.5 and about 2.25 mmole per equivalent of NH₂. The pH ofthe solution generally ranges between about 8 and about 13, preferablyranging between about 10 and about 12.

Thereafter, an amino modifying agent is added to the amino acid solutionwhile stirring. The temperature of the mixture is maintained at atemperature generally ranging between about 5° C. and about 70° C.,preferably between about 10° C. and about 40° C., for a period rangingbetween about 1 and about 4 hours. The amount of amino modifying agentemployed in relation to the quantity of amino acids is based on themoles of total free NH₂ in the amino acids. In general, the aminomodifying agent is employed in an amount ranging between about 0.5 andabout 2.5 mole equivalents, preferably between about 0.75 and about 1.25equivalents, per molar equivalent of total NH₂ groups in the aminoacids.

The reaction is quenched by adjusting the pH of the mixture with asuitable acid, e.g., concentrated hydrochloric acid, until the pHreaches between about 2 and about 3. The mixture separates on standingat room temperature to form a transparent upper layer and a white oroff-white precipitate. The upper layer is discarded and modified aminoacids are collected from the lower layer by filtration or decantation.The crude modified amino acids are then dissolved in water at a pHranging between about 9 and about 13, preferably between about 11 andabout 13. Insoluble materials are removed by filtration and the filtrateis dried in vacuo. The yield of modified amino acids generally rangesbetween about 30 and about 60%, and usually about 45%.

If desired, amino acid esters, such as, for example methyl or ethylesters of amino acids, may be used to prepare the modified amino acidsof the invention. The amino acid esters, dissolved in a suitable organicsolvent such as dimethylformamide or pyridine, are reacted with theamino modifying agent at a temperature ranging between about 5° C. andabout 70° C., preferably about 25° C., for a period ranging betweenabout 7 and about 24 hours. The amount of amino modifying agents usedrelative to the amino acid esters are the same as described above foramino acids.

Thereafter, the reaction solvent is removed under negative pressure andthe ester functionality is removed by hydrolyzing the modified aminoacid ester with a suitable alkaline solution, e.g. 1N sodium hydroxide,at a temperature ranging between about 50° C. and about 80° C.,preferably about 70° C., for a period of time sufficient to hydrolyzeoff the ester group and form the modified amino acid having a freecarboxyl group. The hydrolysis mixture is then cooled to roomtemperature and acidified, e.g. aqueous 25% hydrochloric acid solution,to a pH ranging between about 2 and about 2.5. The modified amino acidprecipitates out of solution and is recovered by conventional means suchas filtration or decantation.

The modified amino acids may be purified by recrystallization or byfractionation on solid column supports. Suitable recrystallizationsolvent systems include acetonitrile, methanol and tetrahydrofuran.Fractionation may be performed on a suitable solid column supports suchas alumina, using methanol/n-propanol mixtures as the mobile phase;reverse phase column supports using trifluoroacetic acid/acetonitrilemixtures as the mobile phase; and ion exchange chromatography usingwater as the mobile phase. When anion exchange chromatography isperformed, a subsequent 0–500 mM sodium chloride gradient is employed.The modified amino acids may also be purified by extraction with a loweralcohol such as methanol, butanol, or isopropanol to remove lowmolecular weight non-sphere making material.

Suitable modified amino acid derivatives include, but are not limitedto, N-cyclohexanoyl-Phe aldehyde, N-acetyl-Phe-aldehyde, N-acetyl-Tyrketone, N-acetyl-Lys ketone and N-acetyl-Leu ketone. Special mention ismade of the modified amino acid derivative N-cyclohexanoyl phenylalaninealdehyde.

Special mention is made of compositions in which the biologically-activeagent includes, calcitonin and the carrier includes acetyl phenylalaninealdehyde, carbomethoxy phenylalanylleucine and acetyl-Phe-Leu-Leualdehyde.

Special mention is also made of a composition which includes 1.5 μg/mlof the biologically-active agent calcitonin and the carrier includes 132mg/ml of acetyl phenylalanine, 33 mg/ml of carbomethoxyphenylalanylleucine, and 25 mg/ml of acetyl-Phe-Leu-Leu-Arg aldehyde.

In one embodiment, the modified and/or modified derivatized amino acidsmay be used directly as a delivery carrier by simply mixing the carrierwith the active ingredient prior to administration. In an alternativeembodiment, the modified amino acids may be used to form microspherescontaining the active agent. The modified and/or modified derivatizedamino acids of the invention are particularly useful for the oraladministration of certain pharmacological agents, e.g., small peptidehormones, which, by themselves, do not pass or only pass slowly throughthe gastro-intestinal mucosa and/or are susceptible to chemical cleavageby acids and enzymes in the gastro-intestinal tract.

If the modified amino acids are to be converted into microspheres, themixture is optionally heated to a temperature ranging between about 20and about 50° C., preferably about 40° C., until the modified aminoacid(s) dissolve. The final solution contains between from about 1 mgand about 2000 mg of modified amino acids per mL of solution, preferablybetween about 1 and about 500 mg per mL. The concentration of activeagent in the final solution varies and is dependent on the requireddosage for treatment. When necessary, the exact concentration can bedetermined by, for example, reverse phase HPLC analysis.

When the modified amino acids are used to prepare microspheres, anotheruseful procedure is as follows: Modified amino acids are dissolved indeionized water at a concentration ranging between about 75 and about200 mg/ml, preferably about 100 mg/ml at a temperature between about 25°C. and about 60° C., preferably about 40° C. Particulate matterremaining in the solution may be removed by conventional means such asfiltration.

Thereafter, the modified amino acid solution, maintained at atemperature of about 40° C., is mixed 1:1 (V/V) with an aqueous acidsolution (also at about 40° C.) having an acid concentration rangingbetween about 0.05 N and about 2 N, preferably about 1.7 N. Theresulting mixture is further incubated at 40° C. for a period of timeeffective for microsphere formation, as observed by light microscopy. Inpracticing this invention, the preferred order of addition is to add themodified amino acid solution to the aqueous acid solution.

Suitable acids for microsphere formation include any acid which does not

-   -   (a) adversely effect the modified amino acids, e.g., initiate or        propagate chemical decomposition;    -   (b) interfere with microsphere formation;    -   (c) interfere with microsphere encapsulation of the cargo; and    -   (d) adversely interact with the cargo.

Preferred acids for use in this invention include acetic acid, citricacid, hydrochloric acid, phosphoric acid, malic acid and maleic acid.

In practicing the invention, a microsphere stabilizing additive may beincorporated into the aqueous acid solution or into the amino acidsolution prior to the microsphere formation process. With some drugs thepresence of such additives promotes the stability and/or dispersibilityof the microspheres in solution.

The stabilizing additives may be employed at a concentration rangingbetween about 0.1 and 5% (w/v), preferably about 0.5% (w/v). Suitable,but non-limiting, examples of microsphere stabilizing additives includegum acacia, gelatin, methyl cellulose, polyethylene glycol, andpolylysine. The preferred stabilizing additives are gum acacia, gelatinand methyl cellulose.

Under the above conditions, the modified amino acid molecules formhollow or solid matrix type microspheres wherein the cargo isdistributed in a carrier matrix or capsule type microspheresencapsulating liquid or solid cargo. If the modified amino acidmicrospheres are formed in the presence of a soluble material, e.g., apharmaceutical agent in the aforementioned aqueous acid solution, thismaterial will be encapsulated within the microspheres. In this way, onecan encapsulate pharmacologically active materials such as peptides,proteins, and polysaccharides as well as charged organic molecules,e.g., antimicrobial agents, which normally have poor bioavailability bythe oral route. The amount of pharmaceutical agent which may beencapsulated by the microsphere is dependent on a number of factorswhich include the concentration of agent in the encapsulating solution,as well as the affinity of the cargo for the carrier.

The modified amino acid microspheres of the invention arepharmacologically harmless and do not alter the physiological andbiological properties of the active agent. Furthermore, theencapsulation process does not alter the pharmacological properties ofthe active agent Any pharmacological agent can be encapsulated withinthe amino acid microspheres. The system is particularly advantageous fordelivering chemical or biological agents which otherwise would bedestroyed or rendered less effective by conditions encountered withinthe body of the animal to which it is administered, before themicrosphere reaches its target zone (i.e., the area in which thecontents of the microsphere are to be released) and pharmacologicalagents which are poorly absorbed in the gastro-intestinal tract. Thetarget zones can vary depending upon the drug employed.

The particle size of the microsphere plays an important role indetermining release of the active agent in the targeted area of thegastro-intestinal tract. The preferred microspheres have diametersbetween about ≦0.1 microns and about 10 microns, preferably betweenabout 0.5 microns and about 5 microns. The microspheres are sufficientlysmall to release effectively the active agent at the targeted areawithin the gastrointestinal tract. Small microspheres can also beadministered parenterally by being suspended in an appropriate carrierfluid (e.g., isotonic saline) and injected directly into the circulatorysystem, intramuscularly or subcutaneously. The mode of administrationselected will vary, of course, depending upon the requirement of theactive agent being administered. Large amino acid microspheres (>50microns) tend to be less effective as oral delivery systems.

The size of the microspheres formed by contacting modified amino acidwith water or an aqueous solution containing active agents can becontrolled by manipulating a variety of physical or chemical parameters,such as the pH, osmolarity or ionic strength of the encapsulatingsolution, size of the ions in solution and by the choice of acid used inthe encapsulating process.

Typically, the pharmacological compositions of the present invention areprepared by mixing an aqueous solution of the carrier with an aqueoussolution of the active ingredient, just prior to administration.Alternatively, the carrier and biologically active ingredient can beadmixed during the manufacturing process. The solutions may optionallycontain additives such as phosphate buffer salts, citric acid, aceticacid, gelatin and gum acacia.

In practicing the invention, stabilizing additives may be incorporatedinto the carrier solution. With some drugs, the presence of suchadditives promotes the stability and dispersibility of the agent insolution.

The stabilizing additives may be employed at a concentration rangingbetween about 0.1 and 5% (W/V), preferably about 0.5% (W/V). Suitable,but non-limiting, examples of stabilizing additives include gum acacia,gelatin, methyl cellulose, polyethylene glycol, and polylysine. Thepreferred stabilizing additives are gum acacia, gelatin and methylcellulose.

The amount of active agent in the composition typically is apharmacologically or biologically effective amount. However, the amountcan be less than a pharmacologically or biologically effective amountwhen the composition is used in a dosage unit form, such as a capsule, atablet or a liquid, because the dosage unit form may contain amultiplicity of carrier/biologically-active agent compositions or maycontain a divided pharmacologically or biologically effective amount.The total effective amounts will be administered by cumulative unitscontaining in total pharmacologically or biologically active amounts ofbiologically-active agent.

The total amount of biologically-active agent to be used can bedetermined by those skilled in the art. However, it has surprisinglybeen found that with certain biologically-active agents, such ascalcitonin, the use of the presently disclosed carriers providesextremely efficient delivery. Therefore, lower amounts ofbiologically-active agent than those used in prior dosage unit forms ordelivery systems can be administered to the subject, while stillachieving the same blood levels and therapeutic effects.

The amount of carrier in the present composition is a delivery effectiveamount and can be determined for any particular carrier orbiologically-active agent by methods known to those skilled in the art.

Dosage unit forms can also include any of excipients; diluents;disintegrants; lubricants; plasticizers; colorants; and dosing vehicles,including, but not limited to water, 1,2-propane diol, ethanol, oliveoil, or any combination thereof.

Administration of the present compositions or dosage unit forms is oralor by intraduodenal injection.

EXAMPLES

The invention will now be illustrated in the following non-limitingexamples which are illustrative of the invention but are not intended tolimit the scope of the invention.

Example 1 Preparation of N-Cyclohexanoylphenylalanine Aldehyde

Phenylalanine methyl ester (1 g., 0.0046 moles) was dissolved inpyridine 5 mL. Cyclohexanoyl chloride (0.62 mL) was added and themixture was stirred for 2 hours. The reaction mixture was poured ontohydrochloric acid (1N) and crushed ice. The aqueous mixture wasextracted twice with toluene. The combined toluene extracts wereconcentrated in vacuo to give 1.1 g of crudeN-cyclohexan-oylphenylalanine methyl ester.

N-Cyclohexanoylphenylalanine methyl ester (0.5 g) was dissolved inethylene glycol dimethyl ether (20 mL). The solution was cooled to −70°C. and diisobutylaluminum hydride (2.04 mL of a 1.5M solution intoluene) was added. The resulting reaction mixture was stirred at −70°C. for 2 hours. The reaction was quenched by dropwise addition of 2Nhydrochloric acid. The mixture was extracted with cold ethyl acetate.The ethyl acetate solution was washed with brine and dried over sodiumsulfate. Concentration in vacuo furnished a white solid which waspurified by silica gel chromatography. ¹H NMR(300 MHz, DMSO-d6): 9.5 (s,1H), 8.2 (d, 1H), 7.2 (m, 5H), 4.2 (m, 1H), 3.2 (d, 1H), 2.7 (d, 1H),2.1 (m, 1H), 1.6 (br. m, 4H), 1.2 (br. m, 6H).

IR (KBr): 3300, 3050, 2900, 2850, 2800, 1700, 1600, 1500 cm-¹. MassSpec.: M+1 m/e 261.

Example 2 Preparation of N-Acetylphenylalanine Aldehyde

N-Acetylphenylalanine methyl ester (4.2 g, 19 mmol) was dissolved inethylene glycol dimethyl ether. The solution was cooled to −70° C. anddiisobutylaluminum hydride (25.3 mL of a 1.5M solution in toluene, 39mmol) was added. The resulting reaction mixture was stirred at −70° C.for 2 hours. The reaction was quenched by addition of 2N hydrochloricacid. The mixture was extracted 4 times with cold ethyl acetate and 4times with toluene. The extracts were combined, washed with brine anddried over magnesium sulfate. Concentration in vacuo followed by silicagel chromatography furnished 2.7 g of a white solid. The NMR wasidentical to that reported in the literature, Biochemistry, 1979, 18,921–926.

Example 3 Preparation of 3-Acetamido-4-(p-Hydroxy)Phenyl-2-Butanone(N-Acetyltyrosinone)

A mixture of tyrosine (28.9 g, 16 mmol), acetic anhydride (97.9 g,96mmol) and pyridine (35 g, 16 mmol) were heated to 100° C. for 1 hour.The reaction mixture was concentrated in vacuo to furnish a yellow oil.The oil was distilled at reduced pressure to furnish 29.9 g or an oil.

¹H NMR (DMSO-d6): NMR (d6-DMSO); 8.2 (d, 1H), 7.3 (d, 2H), 7.0 (d, 2H),4.4 (m, 1H), 3.1 (dd, 1H), 2.7 (dd, 1H), 2.3 (s, 3H), 1.8 (s, 3H).

Example 4 Preparation of 3-Acetamido-7-Amino-2-Butanone(N-Acetyllysinone)

Following the procedure of Example 3 lysine was converted toN-acetyllysinone.

¹H NMR (DMSO-d6): 8.1 (d, 1H), 7.8 (br.m. 1H), 4.1 (m, 1H), 3.0 (m, 2H),2.0 (s, 3H), 1.9 (s, 3H) and 1.3 (br.m, 6H).

Example 5 Preparation of 3-Acetamido-5-Methyl-2-Butanone(N-Acetylleucinone)

Following the procedure of Example 3 leucine was converted toN-acetylleucinone.

¹H NMR (DMSO-d6): 8.1 (d, 1H), 4.2 (m, 1H), 2.0 (s, 3H), 1.8 (s, 3H),0.8 (d, 6H).

Example 6 Modification of 4-(4-Aminophenyl)Butyric Acid Using BenzeneSulfonyl Chloride

4-(4-Aminophenyl)butyric acid, (20 g 0.11 moles) was dissolved in 110 mLof aqueous 2N sodium hydroxide solution. After stirring for about 5minutes at room temperature, benzene sulfonyl chloride (14.2 mL, 0.11moles) was added dropwise into the amino acid solution over a 15 minuteperiod. After stirring for about 3 hours at room temperature the mixturewas acidified to pH 2 by addition of hydrochloric acid. This furnished alight brown precipitate which was isolated by filtration. Theprecipitate was washed with warm water and dried. The yield of4-(phenyl-sulfonamido)4-phenylbutyric acid was 24.3 g (69%). The meltingpoint was 123–25° C.

If necessary, the modified amino acids can be purified byrecrystallization and/or chromatography.

Example 7 Modification of 4-Aminobenzoic Acid Using Benzene SulfonylChloride

Following the procedure of Example 6 4-aminobenzoic acid was convertedto 4-(phenylsulfonamido)benzoic acid.

Example 8 Modification of 4-Aminophenylacetic Acid, 4-AminohippuricAcid, and 4-Aminomethylbenzoic Acid Using Benzene Sulfonyl Chloride

Following the procedure of Example 6, 4-aminophenylacetic acid,4-aminohippuric acid, and 4-amino-methylbenzoic acid were converted to4-(phenylsulfonamido)-phenylacetic acid, 4-(phenylsulfonamido)hippuricacid, and 4-(phenylsulfonamidomethyl)benzoic acid respectively.

Example 9 Modification of Amino Acids with Benzene Sulfonyl Chloride

A mixture of sixteen amino acids were prepared prior to chemicalmodification. The constituents of the mixture are summarized in Table 1.65 grams of the amino acid mixture (total concentration of [−NH₂]groups=0.61 moles) was dissolved in 760 mL of 1N sodium hydroxidesolution (0.7625 equivalents) at room temperature. After stirring for 20minutes, benzene sulfonyl chloride (78 ml, 1 eq.) was added over a 20minute period. The reaction mixture was then stirred for 2.5 hours,without heating. As some precipitation had occurred, additional NaOHsolution (2N) was added to the solution until it reached pH 9.3. Thereaction mixture stirred overnight at room temperature. Thereafter, themixture was acidified using dilute hydrochloric acid (38%, 1:4) and acream colored material precipitated out. The resulting precipitate wasisolated by decantation and dissolved in sodium hydroxide (2N). Thissolution was then reduced in vacuo to give a yellow solid, which wasdried on the lyophilizer.

TABLE 1 Amino Acid Composition No. of moles Weight % of Total of eachAmino No. of Moles Amino Acid (g) Weight Acid (× 10⁻²) of - [—NH₂] Thr2.47 3.8 2.07 2.07 Ser 2.25 3.46 2.1 2.1 Ala 4.61 7.1 5.17 5.17 Val 4.396.76 3.75 3.75 Met 0.53 0.82 0.35 0.35 Ile 2.47 3.8 0.36 0.36 Leu 3.865.94 2.95 2.95 Tyr 1.03 1.58 0.56 0.56 Phe 4.39 6.76 0.27 0.27 His 2.473.8 1.6 3.2 Lys 4.94 7.6 3.4 6.8 Arg 5.13 7.9 2.95 5.90 Glutamine 9.8715.18 6.76 13.42 Glutamic 9.87 15.18 6.70 6.70 Acid Asparagine 3.32 5.112.51 5.02 Aspartic 3.32 5.11 2.50 2.50 Acid

Example 10 Modification of a Mixture of Five Amino Acids Using BenzeneSulfonyl Chloride

An 86.1 g (0.85 moles of NH₂) mixture of amino acids (see Table 2) wasdissolved in 643 mL (1.5 eq.) of aqueous 2N sodium hydroxide solution.After stirring for 30 minutes at room temperature, benzene sulfonylchloride (108 mL, 0.86 moles) was added portionwise into the amino acidsolution over a 15 minute period. After stirring for 2.5 hours at roomtemperature, the pH of the reaction mixture (pH 5) was adjusted to pH 9with additional 2N sodium hydroxide solution. The reaction mixturestirred overnight at room temperature. Thereafter, the pH of thereaction mixture was adjusted to pH 2.5 by addition of dilute aqueoushydrochloric acid solution (4:1, H₂O:HCl) and a precipitate of modifiedamino acids formed. The upper layer was discarded and the resultingyellow precipitate was isolated by decantation, washed with water anddissolved in 2N sodium hydroxide (2N). The solution was reduced in vacuoto give a yellow solid which was lyophilized overnight. The yield ofcrude modified amino acid was 137.9 g.

TABLE 2 Moles of Amino Acid Moles of Amino Acid (× 10⁻²) [—NH₂] × 10⁻²Valine 7.5 7.5 Leucine 10.7 10.5 Phenylalanine 13.4 13.4 Lysine 21.042.0 Arginine 6.0 12.0

Example 11 Modification of a Mixture of Five Amino Acids Using BenzoylChloride

An 86 g (0.85 moles of NH₂) mixture of amino acids (see Table 2 inExample 10) was dissolved in 637 mL (1.5 eq.) of aqueous 2N sodiumhydroxide solution. After stirring for 10 minutes at room temperature,benzoyl chloride (99 mL, 0.85 moles) was added portionwise into theamino acid solution over a 10 minute period. After stirring for 2.5hours at room temperature, the pH of the reaction mixture (pH 12) wasadjusted to pH 2.5 using dilute hydrochloric acid (4:1, H₂O:HCl) and aprecipitate of modified amino acids formed. After settling for 1 hour,the resulting precipitate was isolated by decantation, washed with waterand dissolved in sodium hydroxide (2N). This solution was then reducedin vacuo to give crude modified amino acids as a white solid (220.5 g).

Example 12 Modification of L-Valine Using Benzene Sulfonyl Chloride

L-Valine (50 g, 0.43 mol) was dissolved in 376 mL (0.75 eq.) of aqueous2N sodium hydroxide by stirring at room temperature for 10 minutes.Benzene sulfonyl chloride (68.7 mL, 0.38 mol, 1.25 eq.) was then addedto the amino acid solution over a 20 minute period at room temperature.After stirring for 2 hours at room temperature, a precipitate appeared.The precipitate was dissolved by adding 200 mL of additional 2N sodiumhydroxide solution. After stirring for an additional 30 minutes, diluteaqueous hydrochloric acid solution (4:1, H₂O:HCl) was added until the pHof the reaction mixture reached 2.6. A precipitate of modified aminoacids formed and was recovered by decantation. This material wasdissolved in 2N sodium hydroxide and dried in vacuo to give a whitesolid. Yield of crude modified amino acids=84.6 g, 77%).

Example 13 Modification of Phenylalanine Methyl Ester Using HippurylChloride

L-Phenylalanine Methyl Ester Hydrochloride (15 g, 0.084 mole) wasdissolved in dimethylformamide (DMF) (100 mL) and to this was addedpyridine (30 mL). A solution of hippuryl chloride (16.6 g, 0084 moles in100 mL DMF) was immediately added to the amino acid ester solution intwo portions. The reaction mixture was stirred at room temperatureovernight. The reaction mixture was then reduced in vacuo and dissolvedin 1N aqueous sodium hydroxide. The solution was heated at 70° C. for 3hours in order to hydrolyze the methyl ester to a free carboxyl group.Thereafter, the solution was acidified to pH 2.25 using dilute aqueoushydrochloric acid solution (1:3 HCl/H₂O). A gum-like precipitate formedand this was recovered and dissolved in 1N sodium hydroxide. Thesolution was reduced in vacuo to afford 18.6 g of crude modified aminoacid product (Yield 18.6 g). After recrystallization from acetonitrile,pure modified phenylalanine (12 g) was recovered as a white powder. m.p.223–225° C.

Example 14 Preparation of Dosing Solutions

In a test tube 568 mg of acetyl phenylalanine aldehyde, 132 mg ofcarbomethoxy phenylalanylleucine and 100 mg acetyl-Phe-Leu-Leu-Argaldehyde were added to 2.9 ml of 15% ethanol. The solution was stirredand NaOH (1.0 N) was added to raise the pH to 7.2. Water was added tobring the total volume to 4.0 mL. The sample had a carrier concentrationof 200 mg/mL. Calcitonin (6 μg) was added to the solution. The totalcalcitonin concentration was 1.5 μg/mL.

Following a similar procedure a second solution having 668 mg of acetylphenylalanine aldehyde and 132 mg of carbomethoxy phenylalanylleucine asthe carrier composition and a third solution having as the carrieracetyl phenyl-alanine aldehyde. Each solution had a calcitoninconcentration of 1.5 μg/mL.

Example 15 Preparation of Modified Amino Acid/Salmon CalcitoninCompositions

(a) Preparation of Modified Amino Acid Microspheres ContainingEncapsulated Salmon Calcitonin

The modified amino acid mixture, prepared in accordance with Example 9,was dissolved at 40° C. in distilled water (pH 7.2) at a concentrationof 100 mg/ml. The solution was then filtered with a 0.2 micron filterand the temperature was maintained at 40° C. Salmon calcitonin (SandozCorp., Basil, Switzerland) was dissolved in an aqueous solution ofcitric acid (1.7N) and gelatin (5%) at a concentration of 150 mg/ml.This solution was then heated to 40° C. The two heated solutions werethen mixed 1:1 (v/v). The resulting microsphere suspension was thenfiltered with glass wool and centrifuged for 50 minutes at 1000 g. Thepellet was resuspended with 0.85N citric acid to a volume 5 to 7 foldless than the original volume. Salmon calcitonin concentration of theresuspended pellet was determined by HPLC. Additional microspheres weremade according to the above procedure without salmon calcitonin. These“empty microspheres” were used to dilute the encapsulated salmoncalcitonin microsphere preparation to a final dosing suspension foranimal testing.

(b) Preparation of a Soluble Modified Amino Acid Carrier/SalmonCalcitonin System

A soluble amino acid dosing preparation containing salmon calcitonin wasprepared by dissolving the modified amino acid material in distilledwater (pH 8) to an appropriate concentration. The solution was heated to40° C. and then filtered with a 0.2 micron filter. Salmon calcitonin,also dissolved in distilled water, was then added to the modified aminoacid solution prior to oral administration.

Example 16 In Vivo Experiments in Rats

For each sample six fasted rats were anesthetized. The rats wereadministered, by oral gavage, one of the calcitonin/carrier dosagesprepared in Example 15. The calcitonin concentration in each sample was1.5 μg/ml. Each rat was administered a dosage of two (2) mL/kg each.Blood samples were collected serially from the tail artery. Serumcalcium was determined by testing with a Demand™ Calcium Kit (availablefrom Sigma Chemical Company, St. Louis, Mo., USA). The results of thetest are illustrated in FIG. 1.

Example 17

Three samples having 400 mg/kg of acetyl-Leu aldehyde and 10 μg/kg ofcalcitonin, 400 mg/kg of acetyl-Phe aldehyde and 10 μg/kg of calcitonin,200 mg/kg of acetyl-Leu aldehyde, 200 mg/kg of acetyl-Phe aldehyde and10 μg/kg of calcitonin, respectively were prepared. The samples weregiven to fasted rats as described in Example 16. The results of the testare illustrated graphically in FIG. 2.

Example 18

Two samples having 350 mg/kg of acetyl-Phe aldehyde, 50 mg/kg ofcarbomethoxy-Phe-Leu-OH and 3 μg/kg of calcitonin, 400 mg/kg ofacetyl-Phe aldehyde, 50 mg/kg of carbomethoxy-Phe-Leu-OH and 10 μg/kg ofcalcitonin, respectively were prepared. The samples were given to fastedrats as described in Example 16. The results of the test are illustratedin FIG. 3.

Example 19

Three samples having 284 mg/kg of acetyl-Phe aldehyde and 66 mg/kgacetyl-Leu-Leu-Arg aldehyde, 50 mg/kg of carbomethoxy-Phe-Leu-OH and 3μg/kg of calcitonin in propylene glycol, 284 mg/kg of acetyl-Phealdehyde and 66 mg/kg acetyl-Leu-Leu-Arg aldehyde, 50 mg/kg ofcarbomethoxy -Phe-Leu-OH and 3 μg/kg of calcitonin and 3 μg/kg ofcalcitonin, in aqueous ethanol, respectively were prepared. The sampleswere given to fasted rats as described in Example 16. The results of thetest are illustrated in FIG. 4.

Example 20

Three samples having 400 mg/kg of 4-(phenylsulfon-amido)-4-phenylbutyricacid and 1.5 μg/kg of calcitonin in propylene glycol, 200 mg/kg of4-(phenylsulfonamido)-4-phenylbutyric acid, 200 mg/kg of acetyl-Phealdehyde and 1.5 μg/kg of calcitonin in aqueous ethanol, respectivelywere prepared. The samples were given to fasted rats by intraduodenalinjection. The results of the test are illustrated in FIG. 5.

Example 21

Samples having 600 mg/kg of acetyl-Phe aldehyde and 10 μg/kg ofcalcitonin in aqueous ethanol, and 3 μg/kg of calcitonin, 200 mg/kg ofacetyl-Phe aldehyde, 200 mg/kg N-acetyllysinone, 200 mg/kg acetyl-Leualdehyde and 10 μg/kg of calcitonin were prepared. The samples weregiven to fasted rats as described in Example 16. The results of the testare illustrated in FIG. 6.

Example 22

Three samples having 200 mg/kg of acetyl-Phe aldehyde and 3 μg/kg ofcalcitonin, in aqueous ethanol, dimethyl sulfoxide (DMSO), and oliveoil, respectively, were prepared. Additionally a sample of 3 μg/kg ofcalcitonin in DMSO alone was prepared. The samples were given to rats byintraduodenal injection. The results of the test are illustrated in FIG.7.

Example 23

A sample having 400 mg/kg of cyclohexanoyl-Phe aldehyde and 3 μg/kg ofcalcitonin in aqueous ethanol was prepared. The sample was given tofasted rats as described in Example 16. The results of the test areillustrated in FIG. 8.

Example 24

In vivo evaluation of modified amino acid microspheres containingencapsulated calcitonin and soluble modified amino acidcarrier/calcitonin system, prepared as described in Example 16, wereevaluated in rats. Rats were gavaged with the oral dosing preparationsand blood samples were withdrawn at various time intervals for serumcalcium concentration determinations.

Nine rats are divided into three groups as follows:

-   1. calcitonin microspheres: 10 ug calcitonin/kg body weight by oral    gavage (3 rats);-   2. calcitonin microspheres: 30 ug calcitonin/kg body weight by oral    gavage (3 rats); and-   3. soluble modified amino acid/calcitonin system: 30 ug    calcitonin/kg body weight by oral gavage (3 rats). The rats were    pre-dosed with 0.7 meq of aqueous sodium bicarbonate solution prior    to administration of the soluble system.

Oral gavage dosing of rats is performed. Calcitonin microspheres areprepared immediately prior to dosing and Group 1 rats and Group 2 ratseach receive an appropriate dosage of the microsphere suspension. Group3 rats receives the unencapsulated calcitonin/modified amino acidsystem. Approximately 0.5 ml of blood is withdrawn from each rat justprior to dosing (“0” time) and 1 h, 2 h and 3 h post-dosing. Serum fromthe blood samples are stored at −20° C.

The calcium levels of thawed serum taken from group 1–3 rats areanalyzed by conventional methods. Experimental results in rats havedemonstrated a significant increase in pharmacological activity (i.e.,decreasing serum calcium levels) when calcitonin is orally administeredeither as a encapsulate in modified amino acid microspheres or a mixturewith modified amino acids as compared to basal levels. As shown in FIG.9, soluble modified amino acid solution containing salmon calcitonindemonstrated a significant increase in pharmacological activity (i.e.,decreasing serum calcium levels) when compared to basal levels afteroral administration.

Example 25

Two samples having 366 mg/kg of acetyl-Phe aldehyde, 33 mg/kg ofactinonin and 10 μg/kg of calcitonin, 366 mg of acetyl Phe aldehyde, 33mg/kg of carbomethoxy-Phe-Leu-OH and 10 μg/kg of calcitonin,respectively, were prepared. The samples were given to fasted rats asdescribed in Example 14. The results of the test are illustrated in FIG.10.

Example 26

Two samples having 400 mg/kg of 4-(phenylsulfonamido)-4-phenylbutyricacid and 3 μg/kg of calcitonin, 400 mg/kg of4-(phenylsulfonamido)-4-phenylbutyric acid and 10 μg/kg of calcitonin,respectively were prepared. The samples were given to fasted rats asdescribed in Example 14. The results of the test are illustrated in FIG.11.

Example 27

Two samples having 400 mg/kg of 3-(phenylsulfonamido)benzoic acid and 10μg/kg of calcitonin, 400 mg/kg of 4-(phenylsulfonamido)hippuric acid and10 μg/kg of calcitonin, respectively were prepared. The samples weregiven to fasted rats as described in Example 14. The results of the testare illustrated in FIG. 12.

Example 28

Two samples having 400 mg/kg of 4-(phenylsulfonamido)-4-phenylbutyricacid and 10 μg/kg of calcitonin, 400 mg/kg of4-(phenylsulfonamido)benzoic acid and 10 μg/kg of calcitonin,respectively were prepared. The samples were given to fasted rats asdescribed in Example 14. The results of the test are illustrated in FIG.13.

Example 29

Two samples having 400 mg/kg of 4-(phenylsulfona-mido)-4-phenylbutyricacid and 10 μg/kg of calcitonin, 400 mg/kg of4-(phenylsulfonamido)phenylacetic acid and 10 μg/kg of calcitonin,respectively were prepared. The samples were given to fasted rats asdescribed in Example 14. The results of the test are illustrated in FIG.14.

In Vivo Evaluation of Interferon Preparations in Rats

Following the procedure described herein samples containing the carriersof the subject invention, in a Trizma® hydrochloride buffer solution(Tris-HCl) at a pH of about 7–8, and interferon α2b were prepared. Theanimals were administered the drug by oral gavage. The delivery wasevaluated by using an ELISA assay for human interferon α.

Example 30

A sample having 800 mg/kg of 4-(phenylsulfona -mido)-4-phenylbutyricacid in a buffered solution and 1000 μg/kg of interferon α2b wasprepared. The sample was given to fasted rats as described in Example14. The results of the test are illustrated in FIG. 15.

Example 31

A sample having 400 mg/kg of 4-(phenylsulfonamido -methyl)benzoic acidin a buffered solution and 1000 μg/kg of interferon α2b was prepared.The sample was given to fasted rats as described in Example 14. Theresults of the test are illustrated in FIG. 16.

Example 32

A sample having 800 mg/kg of 4-(phenylsulfona -mido) phenylacetic acidin a buffered solution and 1000 μg/kg of interferon α2b was prepared.The sample was given to fasted rats as described in Example 14. Theresults of the test are illustrated in FIG. 17.

Example 33

A sample having 600 mg/kg of 4-(phenylsulfona -mido)hippuric acid in abuffered solution and 1000 μg/kg of interferon α2b was prepared. Thesample was given to fasted rats as described in Example 14. The resultsof the test are illustrated in FIG. 18.

In Vivo Evaluation of Growth Hormone Preparations in Rats

Following the procedure described herein samples containing the carriersof the subject invention and growth hormone were prepared. The animalswere administered the drug by oral gavage. The delivery was evaluated byusing an ELISA assay for growth hormone.

Example 34

A sample having 1000 mg/kg of 4-(phenylsulfona -mido)-4-phenylbutyricacid and 1 mg/kg of growth hormone was prepared. The sample was given tohypophysectomized rats as described in Example 14. The results of thetest are illustrated in FIG. 19.

Example 35

A sample having 500 mg/kg of 4-(phenylsulfona -mido)-4-phenylbutyricacid and 1 mg/kg of growth hormone was prepared. In a comparison a groupof hypophysectomized rats were given samples of growth hormone without acarrier. The samples were given to hypophysectomized rats as describedin Example 14. The results of the test are illustrated in FIG. 20.

Example 36

Two samples having 500 mg/kg of 4-(phenylsulfona -mido)-4-phenylbutyricacid and 6 mg/kg of growth hormone were prepared. The samples were givento normal rats as described in Example 14. The results of the tests areillustrated in FIG. 21.

In Vivo Evaluation of Cromoglycolate Preparations in Rats

Example 37

Following the procedure described herein samples containing the carriersof the subject invention and disodium cromoglycolate were prepared. Thesample, in 0.85N citric acid and 0.5% acacia, contained 200 mg/kg of4-(phenylsulfonamido)-4-phenylbutyric acid and 50 mg/kg of disodiumcromoglycate. The animals were administered the drug by oral gavage. Thedelivery was evaluated by using the procedure described by A. Yoshimi inPharmcobio-Dyn., 15, pages 681–686, (1992). The results of the tests areillustrated in FIG. 22.

As clearly illustrated by the data in the Examples and Figures the useof compositions of the subject invention show significant advantages forthe delivery of biologically active agents.

All patents, applications, and publications mentioned herein areincorporated by reference herein.

Many variations of the present invention will suggest themselves tothose skilled in the art in light of the above detailed disclosure. Forexample, poly (amino acids) which are formed by a bond other than anamide bond, e.g., an ester or an anhydride linkage, may be derivatizedand modified for use as carriers in accordance with the presentinvention. All such modifications are within the full intended scope ofthe appended claims.

1. A composition comprising: (A) at least one biologically-active agent;and (B) a compound having the formula:Ar—Y—(R¹)_(n)—OH wherein: Ar is a substituted or unsubstituted phenyl; Yis

R¹ has the formula

wherein: R³ is C₁ to C₂₄ alkyl; R⁴ is hydrogen, C₁ to C₄ alkyl, or C₁ toC₄ alkenyl; and n is equal to
 1. 2. The composition of claim 1, whereinsaid biologically-active agent is selected from the group consisting ofa peptide, a polysaccharide, a mucopolysaccharide, a carbohydrate, alipid or any combination thereof.
 3. The composition of claim 2, whereinsaid biologically-active agent is a peptide.
 4. The composition of claim2, wherein said biologically-active agent is a polysaccharide.
 5. Thecomposition of claim 2, wherein said biologically-active agent is amucopolysaccharide.
 6. The composition of claim 1, wherein saidbiologically-active agent is selected from the group consisting of humangrowth hormone, bovine growth hormone, growth hormone-releasing hormone,an interferon, interleukin-I, interleukin-II, insulin, heparin, lowmolecular weight heparin, calcitonin, erythropoietin, atrial natureticfactor, an antigen, a monoclonal antibody, somatostatin,adrenocorticotropin, gonadotropin releasing hormone, oxytocin,vasopressin, cromolyn sodium, vancomycin, desferrioxamine, or anycombination thereof.
 7. The composition of claim 6, wherein saidbiologically-active agent is human growth hormone.
 8. The composition ofclaim 6, wherein said biologically-active agent is interferon.
 9. Thecomposition of claim 6, wherein said biologically-active agent isinsulin.
 10. The composition of claim 6, wherein saidbiologically-active agent is heparin.
 11. The composition of claim 6,wherein said biologically-active agent is low molecular weight heparin.12. The composition of claim 6, wherein said biologically-active agentis calcitonin.
 13. The composition of claim 6, wherein saidbiologically-active agent is erythropoietin.
 14. The composition ofclaim 6, wherein said biologically-active agent is an antigen.
 15. Thecomposition of claim 6, wherein said biologically-active agent iscromolyn sodium.
 16. A dosage unit form comprising (A) the compositionof claim 1; and (B) (a) an excipient (b) a diluent, (c) a disintegrant,(d) a lubricant, (e) a plasticizer, (f) a colorant, (g) a dosingvehicle, or (h) any combination thereof.
 17. The dosage unit form ofclaim 16, comprising a tablet, a capsule, or a liquid.
 18. The dosageunit form of claim 17, wherein said biologically-active agent isselected from the group consisting of a peptide, a polysaccharide, amucopolysaccharide, a carbohydrate, a lipid or any combination thereof.19. The dosage unit form of claim 18, wherein said biologically-activeagent is a peptide.
 20. The dosage unit form of claim 18, wherein saidbiologically-active agent is a polysaccharide.
 21. The dosage unit formof claim 18, wherein said biologically-active agent is amucopolysaccharide.
 22. The dosage unit form of claim 17, wherein saidbiologically-active agent is selected from the group consisting of humangrowth hormone, bovine growth hormone, growth hormone-releasing hormone,an interferon, interleukin-I, interleukin-II, insulin, heparin, lowmolecular weight heparin, calcitonin, erythropoietin, atrial natureticfactor, an antigen, a monoclonal antibody, somatostatin,adrenocorticotropin, gonadotropin releasing hormone, oxytocin,vasopressin, cromolyn sodium, vancomycin, desferrioxamine, or anycombination thereof.
 23. The dosage unit form of claim 22, wherein saidbiologically-active agent is human growth hormone.
 24. The dosage unitform of claim 22, wherein said biologically-active agent is interferon.25. The dosage unit form of claim 22, wherein said biologically-activeagent is insulin.
 26. The dosage unit form of claim 22, wherein saidbiologically-active agent is heparin.
 27. The dosage unit form of claim22, wherein said biologically-active agent is low molecular weightheparin.
 28. The dosage unit form of claim 22, wherein saidbiologically-active agent is calcitonin.
 29. The dosage unit form ofclaim 22, wherein said biologically-active agent is erythropoietin. 30.The dosage unit form of claim 22, wherein said biologically-active agentis an antigen.
 31. The dosage unit form of claim 22, wherein saidbiologically-active agent is cromolyn sodium.
 32. A method for preparinga composition, said method comprising mixing: (A) at least onebiologically-active agent; and (B) a compound having the formula:Ar—Y—(R¹)_(n)—OH wherein: Ar is a substituted or unsubstituted phenyl; Yis

R¹ has the formula

wherein: R³ is C₁ to C₂₄ alkyl, (C₁ to C₁₀ alkyl) phenyl, or phenyl (C₁to C₁₀ alkyl); R⁴ is hydrogen, C₁ to C₄ alkyl, or C₁ to C₄ alkenyl; andn is equal to
 1. 33. The method of claim 32, wherein saidbiologically-active agent is selected from the group consisting of apeptide, a polysaccharide, a mucopolysaccharide, a carbohydrate, a lipidor any combination thereof.
 34. The method of claim 33, wherein saidbiologically-active agent is a peptide.
 35. The method of claim 33,wherein said biologically-active agent is a polysaccharide.
 36. Themethod of claim 33, wherein said biologically-active agent is amucopolysaccharide.
 37. The method of claim 32, wherein saidbiologically-active agent is selected from the group consisting of humangrowth hormone, bovine growth hormone, growth hormone-releasing hormone,an interferon, interleukin-I, interleukin-II, insulin, heparin, lowmolecular weight heparin, calcitonin, erythropoietin, atrial natureticfactor, an antigen, a monoclonal antibody, somatostatin,adrenocorticotropin, gonadotropin releasing hormone, oxytocin,vasopressin, cromolyn sodium, vancomycin, desferrioxamine, or anycombination thereof.
 38. The method of claim 37, wherein saidbiologically-active agent is human growth hormone.
 39. The method ofclaim 37, wherein said biologically-active agent is interferon.
 40. Themethod of claim 37, wherein said biologically-active agent is insulin.41. The method of claim 37, wherein said biologically-active agent isheparin.
 42. The method of claim 37, wherein said biologically-activeagent is low molecular weight heparin.
 43. The method of claim 37,wherein said biologically-active agent is calcitonin.
 44. The method ofclaim 37, wherein said biologically-active agent is erythropoietin. 45.The method of claim 37, wherein said biologically-active agent is anantigen.
 46. The method of claim 37, wherein said biologically-activeagent is cromolyn sodium.